Late blight, caused by the oomycete pathogen Phytophthora infestans is world-wide the most destructive disease for potato cultivation. The disease also threatens the tomato crop. The urgency of obtaining resistant cultivars has intensified as more virulent, crop-specialised and pesticide resistant strains of the pathogen are rapidly emerging.
A way to prevent crop failures or reduced yields is the application of fungicides that prevent or cure an infection by P. infestans. However, the application of crop protectants is widely considered to be a burden for the environment. Thus, in several Western countries, legislation is becoming more restrictive and partly prohibitive to the application of specific fungicides, making chemical control of the disease more difficult. An alternative approach is the use of cultivars that harbour partial or complete resistance to late blight. Two types of resistance to late blight have been described and used in potato breeding. One kind is conferred by a series of major, dominant genes that render the host incompatible with specific races of the pathogen (race specific resistance). Eleven such R genes (R1-R11) have been identified and are believed to have originated in the wild potato species Solanum demissum, which is native to Mexico, where the greatest genetic variation of the pathogen is found. Several of these R genes have been mapped on the genetic map of potato (reviewed in Gebhardt and Valkonen, 2001 Annu. Rev. Phytopathol. 39: 79-102). R1 and R2 are located on chromosomes 5 and 4, respectively. R3, R6 and R7 are located on chromosome 11. Unknown R genes conferring race specific resistance to late blight have also been described in S. tuberosum ssp. andigena and S. berthaultii (Ewing et al., 2000 Mol. Breeding. 6: 25-36). Because of the high level of resistance and ease of transfer, many cultivars contain S. demissum derived resistance. Unfortunately, S. demissum derived race specific resistance, although nearly complete, is not durable. Once newly bred cultivars are grown on larger scale in commercial fields, new virulences emerge in P. infestans that render the pathogen able to overcome the introgressed resistance. The second type of resistance, termed field resistance and often quantitative in nature, is thought to be race non-specific and more durable. Field resistance to late blight can be found in several Mexican and Middle and South American Solanum species (Rossi et al., 1986 PNAS 95:9750-9754).
Diploid S. bulbocastanum from Mexico and Guatemala is one of the tuber bearing species that is known for its high levels of field resistance to late blight (Niederhauser and Mills, 1953 Phytopathology 43: 456-457). Despite differences in endosperm balance numbers, introgression of the S. bulbocastanum resistance trait has been successful. Ploidy manipulations and a series of tedious bridge crosses has resulted in S. bulbocastanum derived, P. infestans resistant germplasm (Hermsen and Ramanna, 1969 Euphytica 18:27-35; 1973 Euphytica 22:457-466; Ramanna and Hermsen, 1971 Euphytica 20:470-481; Hermsen and De Boer, 1971 Euphytica 20:171-180). However, almost 40 years after the first crosses and intense and continuous breeding efforts by potato breeders in the Netherlands with this germplasm, late blight resistant cultivars still remain to be introduced on the market. Successful production of somatic hybrids of S. bulbocastanum and S. tuberosum has also been reported (Thieme et al., 1997 Euphytica 97(2):189-200; Helgeson et al., 1998 Theor Appl. Genet 96:738-742). Some of these hybrids and backcrossed germplasm were found to be highly resistant to late blight, even under extreme disease pressure. Despite reports of suppression of recombination, resistance in the backcrossed material appeared to be on chromosome 8 within an approximately 6 cM interval between the RFLP markers CP53 and CT64 (Naess et al., 2000 Theor. Appl Genet 101:697-704). A CAPS marker derived from the tomato RFLP probe CT88 cosegregated with resistance. Suppression of recombination between the S. bulbocastanum and S. tuberosum chromosomes forms a potential obstacle for successful reconstitution of the recurrent cultivated potato germplasm to a level that could meet the standards for newly bred potato cultivars. Isolation of the genes that code for resistance found in S. bulbocastanum and subsequent transformation of existing cultivars with these genes, would be a much more straight forward and quicker approach when compared to introgression breeding.
The cloning and molecular characterisation of numerous plant R genes conferring disease resistance to bacteria, fungi, viruses, nematodes, and insects has identified several structural features characteristic to plant R genes (reviewed in Dangl and Jones, 2001 Nature 411, 826-833). The majority are members of tightly linked multigene families and all R genes characterised so far, with the exception of Pto, encode leucine-rich repeats (LRRs), structures shown to be involved in protein-protein interactions. LRR containing R genes can be divided into two classes based on the presence of a putative tripartite nucleotide-binding site (NBS). R genes of the NBS-LRR class comprise motifs that are shared with animal apoptosis regulatory proteins (van der Biezen et al., 1998 Curr. Biol. 8, 226-227; Aravind et al., 1999 Trends Biochem. Sci. 24, 47-53) and can be subdivided into two subgroups based on their N-terminal domain, which either exhibits sequence similarity to the Drosophila Toll protein and the mammalian interleukin-1 receptor domain (TIR-NBS-LRR), or contains a potential leucine zipper or coiled-coil domain (CC-NBS-LRR; Pan et al., 2000 Genetics. 155:309-22). LRR R genes without an NBS encode transmembrane proteins, whose extracellular N-terminal region is composed of LRRs (Jones et al., 1994 Adv. Bot. Res. 24, 89-167). These genes can be divided into two subgroups based on the presence of a cytosolic serine/threonine kinase domain (Song et al., 1995 Science, 270, 1804-1806). Four R genes have currently been cloned from potato. All four, including the S. demissum derived R1 gene conferring race specific resistance to late blight, belong to the CC-NBS-LRR class of plant R genes (Bendahmane et al., 1999 Plant Cell 11, 781-791; Bendahmane et al., 2000 Plant J. 21, 73-81; van der Vossen et al., 2000 Plant Journal 23, 567-576; Ballvora et al., 2002 Plant Journal 30, 361-371).
The invention provides an isolated or recombinant nucleic acid comprising a nucleic acid coding for the amino acid sequence of FIG. 8 (SEQ ID NO: 54) or a functional fragment or a homologue thereof. The protein coded by said amino acid has been detected as being member of a cluster of genes identifiable by phylogenetic tree analysis, which thus far consists of the proteins Rpi-blb, RGC1-blb, RGC3-blb and RGC4-blb (herein also called the Rpi-blb gene cluster) of FIG. 9.
Phylogenetic tree analysis is carried out as follows. First a multiple sequence alignment is made of the nucleic acid sequences and/or preferably of the deduced amino acid sequences of the genes to be analysed using CLUSTALW, which is in standard use in the art. ClustalW produces a .dnd file, which can be read by TREEVIEW. The phylogenetic tree depicted in FIG. 9A is a phylogram.
Phylogenetic studies of the deduced amino acid sequences of Rpi-blb, RGC1-blb, RGC3-blb, RGC4-blb and those of the most similar genes from the art (as defined by the BLASTX) derived from diverse species, using the Neighbour-Joining method of Saitou and Nei (1987 Molecular Biology and Evolution 4, 406-425), shows that corresponding genes or functional fragments thereof of the Rpi-blb gene cluster can be placed in a separate branch (FIG. 9A).
Sequence comparisons between the four members of the Rpi-blb gene cluster identified on 8005-8 BAC clone SPB4 show that sequence homology within the Rpi-blb gene cluster varies between 70% and 81% at the amino acid sequence level. The deduced amino acid sequence of Rpi-blb shares the highest overall homology with RGC3-blb (81% amino-acid sequence identity; Table 4). When the different domains are compared it is clear that the effector domains present in the N-terminal halves of the proteins (coiled-coil and NBS-ARC domains) share a higher degree of homology (91% sequence identity) than the C-terminal halves of these proteins which are thought to contain the recognition domains (LRRs; 71% amino acid sequence identity). Comparison of all four amino-acid sequences revealed a total of 104 Rpi-blb specific amino acid residues (FIG. 10). The majority of these are located in the LRR region (80/104). Within the latter region, these specific residues are concentrated in the LRR subdomain xxLxLxxxx (SEQ ID NO: 1). The relative frequency of these specific amino-acid residues within this LRR subdomain is more than two times higher (28.3%) than that observed in the rest of the LRR domain (12.3%). The residues positioned around the two conserved leucine residues in the consensus xxLxxLxxxx (SEQ ID NO: 2) are thought to be solvent exposed and are therefore likely to be involved in creating/maintaining recognition specificity of the resistance protein.
Sequences of additional members of the Rpi-blb gene cluster can be obtained by screening genomic DNA or insert libraries, e.g. BAC libraries with primers based on signature sequences of the Rpi-blb gene. Screening of various Solanum BAC libraries with primer sets A and/or B (Table 2 and FIG. 7) identified numerous Rpi-blb homologues derived from different Solanum species. Alignment of these additional sequences with those presented in FIG. 10 will help identify additional members of the Rpi-blb gene cluster and specific amino acid residues therein responsible for P. infestans resistance specificity. Furthermore, testing additional sequences in the above described phylogenetic tree analyses, e.g. using the Neighbour-Joining method of Saitou and Nei (1987 Molecular Biology and Evolution 4, 406-425), provides additional identification of genes belonging to the Rpi-blb gene cluster.
The invention provides the development of an intraspecific mapping population of S. bulbocastanum that segregated for race non-specific resistance to late blight. The resistance was mapped on chromosome 8, in a region located 0.3 cM distal from CT88. Due to the race non-specific nature of the resistance, S. bulbocastanum late blight resistance has always been thought to be R gene independent. However, with the current invention we demonstrate for the first time that S. bulbocastanum race non-specific resistance is in fact conferred by a gene bearing similarity to an R gene of the NBS-LRR type.
The invention further provides the molecular analysis of this genomic region and the isolation by map based cloning of a DNA-fragment of the resistant parent that harbours an R gene, designated Rpi-blb. This DNA-fragment was subcloned from an approximately 80 kb bacterial artificial chromosome (BAC) clone which contained four complete R gene-like sequences in a cluster-like arrangement. Transformation of a susceptible potato cultivar by Agrobacterium tumefaciens revealed that one of the four R gene-like sequences corresponds to Rpi-blb that provides the race non-specific resistance to late blight. Characterisation of the Rpi-blb gene showed that it is a member of the NBS-LRR class of plant R genes. The closest functionally characterised sequences of the prior art are members of the 12 resistance gene family in tomato. These sequences have an overall amino acid sequence identity of approximately 32% with that of Rpi-blb.
Thus, in a first embodiment, the invention provides an isolated or recombinant nucleic acid, said nucleic acid encoding a gene product having the sequence of Rpi-blb or a functional fragment thereof that is capable of providing a member of the Solanaceae family with race non-specific resistance against an oomycete pathogen.
Isolation of the gene as provided here that codes for the desired resistance trait against late blight and subsequent transformation of existing potato and tomato cultivars with this gene now provides a much more straightforward and quicker approach when compared to introgression breeding. The results provided here offer possibilities to further study the molecular basis of the plant pathogen interaction, the ecological role of R genes in a wild Mexican potato species and are useful for development of resistant potato or tomato cultivars by means of genetic modification.
In contrast to the R genes cloned and described so far, the gene we provide here is the first isolated R gene from a Solanum species that provides race non-specific resistance against an oomycete pathogen. Notably, the invention provides here a nucleic acid wherein said Solanum species that is provided with the desired resistance comprises S. tuberosum. In particular, it is the first gene that has been isolated from a phylogenetically distinct relative of cultivated potato, S. bulbocastanum, for which it was shown by complementation assays, that it is functional in S. tuberosum. These data imply that the gene Rpi-blb can easily be applied in potato production without a need for time-consuming and complex introgression breeding.
The following definitions are provided for terms used in the description and examples that follow.
Nucleic acid: a double or single stranded DNA or RNA molecule.
Oligonucleotide: a short single-stranded nucleic acid molecule.
Primer the term primer refers to an oligonucleotide that can prime the synthesis of nucleic acid.
Homology: homology is the term used for the similarity or identity of biological sequence information. Homology may be found at the nucleotide sequence and/or encoded amino acid sequence level. For calculation of precentage identity the BLAST algorithm can be used (Altschul et al., 1997 Nucl. Acids Res. 25:3389-3402) using default parameters or, alternatively, the GAP algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453), using default parameters, which both are included in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA. BLAST searches assume that proteins can be modelled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, 1993 Comput. Chem. 17:149-163) and XNU (Clayerie and States, 1993 Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination.
As used herein, ‘sequence identity’ or ‘identity’ in the context of two protein sequences (or nucleotide sequences) includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognised that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acids are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percentage sequence identity may be adjusted upwards to correct for the conservative nature of the substitutions. Sequences, which differ by such conservative substitutions are said to have ‘sequence similarity’ or ‘similarity’. Means for making these adjustments are well known to persons skilled in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is give a score of zero, a conservative substitution is given a score between 0 and 1. The scoring of conservative substitutions is calculated, e.g. according to the algorithm of Meyers and Miller (Computer Applic. Biol. Sci. 4:11-17, 1988).
As used herein, ‘percentage of sequence identity’ means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence or nucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid or nucleic acid base residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably the amino acid sequence of the protein of the invention shares at least 82% or higher homology with the sequence as depicted in FIG. 8. As shown in Table 4, the closest functionally characterised sequence of the prior art (members of the I2 Fusarium resistance gene cluster in tomato) has a much lower level of amino acid sequence identity than this (32% with respect to that of Rpi-blb). Homology within the gene cluster of the present invention varies between 70% and 81% at the amino acid sequence level.
Homologous nucleic acid sequences are nucleic acid sequences coding for a homologous protein defined as above. One example of such a nucleic acid is the sequence as provided in FIG. 6A. However, there are many sequences which code for a protein which is 100% identical to the protein as depicted in FIG. 8. This is due to the ‘wobble’ in the nucleotide triplets, where more than one triplet can code for one and the same amino acid. Thus, even without having an effect on the amino acid sequence of the protein the nucleotide sequence coding for this protein can be varied substantially. It is acknowledged that nucleotide sequences coding for amino acid sequences that are not 100% identical to said protein can contain even more variations. Therefore, the percentage identity on nucleic acid sequence level can vary within wider limits, without departing from the invention.
Promoter: the term “promoter” is intended to mean a short DNA sequence to which RNA polymerase and/or other transcription initiation factors bind prior to transcription of the DNA to which the promoter is functionally connected, allowing transcription to take place. The promoter is usually situated upstream (5′) of the coding sequence. In its broader scope, the term “promoter” includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site. Such regulatory sequences are, e.g., sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological conditions. The promoter region should be functional in the host cell and preferably corresponds to the natural promoter region of the Rpi-blb resistance gene. However, any heterologous promoter region can be used as long as it is functional in the host cell where expression is desired. The heterologous promoter can be either constitutive or regulatable, tissue specific or not specific. A constitutive promoter such as the CaMV 35S promoter or T-DNA promoters, all well known to those skilled in the art, is a promoter which is subjected to substantially no regulation such as induction or repression, but which allows for a steady and substantially unchanged transcription of the DNA sequence to which it is functionally bound in all active cells of the organism provided that other requirements for the transcription to take place is fulfilled. It is possible to use a tissue-specific promoter, which is driving expression in those parts of the plant which are prone to pathogen infection. In the case of Phytophthora a promoter which drives expression in the leaves, such as the ferredoxin promoter, can be used. A regulatable promoter is a promoter of which the function is regulated by one or more factors. These factors may either be such which by their presence ensure expression of the relevant DNA sequence or may, alternatively, be such which suppress the expression of the DNA sequence so that their absence causes the DNA sequence to be expressed. Thus, the promoter and optionally its associated regulatory sequence may be activated by the presence or absence of one or more factors to affect transcription of the DNA sequences of the genetic construct of the invention. Suitable promoter sequences and means for obtaining an increased transcription and expression are known to those skilled in the art.
Terminator: the transcription terminator serves to terminate the transcription of the DNA into RNA and is preferably selected from the group consisting of plant transcription terminator sequences, bacterial transcription terminator sequences and plant virus terminator sequences known to those skilled in the art.
Gene: the term “gene” is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5′-upstream and 3′-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5′-upstream or 3′-downstream region. The 5′-upstream region may comprise a regulatory sequence that controls the expression of the gene, typically a promoter. The 3′-downstream region may comprise sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3′ untranslated region. The term “resistance gene” is an isolated nucleic acid according to the invention said nucleic acid encoding a gene product that is capable of providing a plant with resistance against a pathogen, more specifically said plant being a member of the Solanaceae family, more preferably potato or tomato, said pathogen more specifically being an oomycete pathogen, more specifically Phytophthora, more specifically Phytophthora infestans, said nucleic acid preferably comprising a sequence as depicted in FIG. 8 (SEQ ID NO: 54) or part thereof, or a homologous sequence with essentially similar functional and structural characteristics. A functionally equivalent fragment of such a resistance gene or nucleic acid as provided by the invention encodes a fragment of a polypeptide having an amino acid sequence as depicted in FIG. 8 (SEQ ID NO: 54) or part thereof, or a homologous and/or functionally equivalent polypeptide, said fragment exhibiting the characteristic of providing at least partial resistance to an oomycete infection such as caused by P. infestans when incorporated and expressed in a plant or plant cell.
Resistance gene product: a polypeptide having an amino acid sequence as depicted in FIG. 8 (SEQ ID NO: 54) or part thereof, or a homologous and/or functionally equivalent polypeptide exhibiting the characteristic of providing at least partial resistance to an oomycete infection such as caused by P. infestans when incorporated and expressed in a plant or plant cell.
Functionally equivalents of the protein of the invention are proteins that are homologous to and are obtained from the protein depicted in FIG. 8 (SEQ ID NO: 54) by replacing, adding and/or deleting one or more amino acids, while still retaining their pathogen resistance activity. Such equivalents can readily be made by protein engineering in vivo, e.g. by changing the open reading frame capable of encoding the protein so that the amino acid sequence is thereby affected. As long as the changes in the amino acid sequences do not altogether abolish the activity of the protein such equivalents are embraced in the present invention. Further, it should be understood that equivalents should be derivable from the protein depicted in FIG. 8 (SEQ ID NO: 54) while retaining biological activity, i.e. all, or a great part of the intermediates between the equivalent protein and the protein depicted in FIG. 8 should have pathogen resistance activity. A great part would mean 30% or more of the intermediates, preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more.
Preferred equivalents are equivalents in which the leucine rich repeat region is highly homologous to the LRR region as depicted in FIG. 8 (SEQ ID NO: 54). Other preferred equivalents are equivalents wherein the N-terminal effector domain is essential the same as the effector domain of Rpi-blb.
The protein of the invention comprises a distinct N-terminal effector domain and a leucine rich repeat domain. It is believed that conservation of these regions is essential for the function of the protein, although some variation is allowable. However, the other parts of the protein are less important for the function and may be more susceptible to change.
In order to provide a quick and simple test if the modified proteins and/or the gene constructs capable of expressing said modified proteins which are described here or any new constructs which are obvious to the person skilled in the art after reading this application indeed can yield a resistance response the person skilled in the art can perform a rapid transient expression test known under the name of ATTA (Agrobacterium tumefaciens Transient expression Assay). In this assay (of which a detailed description can be found in Van den Ackerveken, G., et al., Cell 87, 1307-1316, 1996) the nucleotide sequence coding for the modified protein which is to be tested is placed under control of the CaMV 35S promoter and introduced into an Agrobacterium strain which is also used in protocols for stable transformation. After incubation of the bacteria with acetosyringon or any other phenolic compound which is known to enhance Agrobacterium T-DNA transfer, 1 ml of the Agrobacterium culture is infiltrated into an in situ plant leaf (from e.g. a tobacco or potato or tomato plant) by injection after which the plants are placed in a greenhouse and infected with a pathogen, preferably P. infestans. After 2-5 days the leaves can be scored for occurrence of resistance symptoms.
In the present invention we have identified and isolated the resistance gene Rpi-blb, which confers race non-specific resistance to Phytophthora infestans. The gene was cloned from a Solanum bulbocastanum genotype that is resistant to P. infestans. The isolated resistance gene according to the invention can be transferred to a susceptible host plant using Agrobacterium mediated transformation or any other known transformation method, and is involved in conferring the host plant resistant to plant pathogens, especially P. infestans. The host plant can be potato, tomato or any other plant, in particular a member of the Solanaceae family that may be infected by such a plant pathogen. The present invention provides also a nucleic acid sequence coding for this protein or a functional equivalent thereof, preferably comprising the Rpi-blb gene, which is depicted in FIG. 6 (SEQ ID NOs: 48, 49, 50, 51, 52 and 53).
With the Rpi-blb resistance protein or functionally equivalent fragment thereof according to the invention, one has an effective means of control against plant pathogens, since the gene coding for the protein can be used for transforming susceptible plant genotypes thereby producing genetically transformed plants having a reduced susceptibility or being preferably resistant to a plant pathogen. In particular, a plant genetically transformed with the Rpi-blb resistance gene according to the invention has a reduced susceptibility to P. infestans. 
In a preferred embodiment the Rpi-blb resistance gene comprises the coding sequence provided in FIG. 6A (SEQ ID NO: 48) or any homologous sequence or part thereof preceded by a promoter region and/or followed by a terminator region. The promoter region should be functional in plant cells, and preferably correspond to the native promoter region of the Rpi-blb gene. However, a heterologous promoter region that is functional in plant cells can be used in conjunction with the coding sequences.
In addition the invention relates to the Rpi-blb resistance protein which is encoded by the Rpi-blb gene according to the invention and which has an amino acid sequence provided in FIG. 8 (SEQ ID NO: 54), or a functional equivalent thereof.
The signal that triggers the expression of the resistance gene in the wild-type S. bulbocastanum or in the transgenic plants of the invention is probably caused by the presence of a pathogen, more specifically the pathogen P. infestans. Such systems are known for other pathogen-plant interactions (Klement, Z., In: Phytopathogenic Prokaryotes, Vol. 2, eds.: Mount, M. S, and Lacy, G. H., New York, Academic Press, 1982, pp. 149-177), and use of this system can be made to increase the applicability of the resistance protein resulting in a resistance to more pathogens (see EP 474 857). This system makes use of the elicitor compound derived from the pathogen and the corresponding resistance gene, wherein the resistance gene when activated by the presence of the elicitor would lead to local cell death (hypersensitive reaction). In case of the present resistance gene, the corresponding elicitor component has not yet been disclosed, but it is believed that this is achievable by a person skilled in the art. Once the elicitor component is isolated it will be possible to transform the gene coding for said elicitor together with the gene coding for the resistance protein into plant, whereby one of the genes is under control of a pathogen-inducible promoter. These promoters are well known in the art (e.g. prp1, Fis1, Bet v 1, Vst1, gstA1, and sesquiterpene cyclase, but any pathogen-inducible promoter which is switched on after pathogen infection can be used). If the transgenic plant contains such a system, then pathogen attack which is able to trigger the pathogen-inducible promoter will cause production of the component which is under control of said promoter, and this, in connection with the other component being expressed constitutively, will cause the resistance reaction to occur.
It will also be possible to mutate the resistance protein causing it to be in an active state (see EP1060257). Since this would permanently result in the resistance reaction to occur, which ultimately leads to local cell death, care should be taken not to constitutively express the resistance protein. This can be accomplished by placing the mutated resistance protein under control of a pathogen-inducible promoter, which not only would allow for expression of the active resistance protein only at times of pathogen attack, but would also allow a broader pathogen range to induce the hypersensitive reaction. Mutation of threonine and serine residues to aspartic acid and glutamic acid residues frequently leads to activation, as was shown in many proteins of which the activity is modulated by phosphorylation, e.g. in a MAPK-activated protein (Engel et al., 1995, J. Biol. Chem. 270, 27213-27221), and in a MAP-kinase-kinase protein (Huang et al., 1995 Mol. Biol. Cell 6, 237-245). Also C- and N-terminal as well as internal deletion mutants of these proteins can be tested for suitable mutants.
A more undirected way of identifying interesting mutants of which constitutive activity is induced is through propagation of the protein-encoding DNA in so-called E. coli ‘mutator’ strains.
A rapid way of testing all made mutants for their suitability to elicit a hypersensitive response is through a so-called ATTA assay (Van den Ackerveken, G., et al., Cell 87, 1307-1316, 1996). Many mutants can be screened with low effort to identify those that will elicit an HR upon expression.
The invention also provides a vector comprising a nucleic acid as provided herein, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae family with resistance against an oomycete pathogen, or a functionally equivalent isolated or recombinant nucleic acid in particular wherein said member comprises S. tuberosum or Lycopersicon esculentum. 
The invention also provides a host cell comprising a nucleic acid or a vector according to the invention. An example of said host cell is provided in the detailed description herein. In a particular embodiment, said host cell comprises a plant cell. As a plant cell a cell derived from a member of the Solanaceae family is preferred, in particular wherein said member comprises S. tuberosum or Lycopersicon esculentum. From such a cell, or protoplast, a transgenic plant, such as transgenic potato plant or tomato plant with resistance against an oomycete infection can arise. The invention thus also provides a plant, or tuber root, fruit or seed or part or progeny derived thereof comprising a cell according to the invention.
Furthermore, the invention provides a proteinaceous substance, exhibiting the characteristic of providing at least partial resistance to an oomycete infection such as caused by P. infestans when incorporated and expressed in a plant or plant cell. In particular such a proteinaceous substance is provided that is encoded by a nucleic acid according to the invention. In a preferred embodiment, the invention provides a proteinaceous substance comprising an amino acid sequence as depicted in FIG. 8 or a functional equivalent thereof. Preferably, such a functional equivalent will comprise one or more sequences which are relatively unique to Rpi-blb in comparison to RGC3-blb, RGC-blb and RGC4-blb. Such sequences can be spotted in the alignment (see FIG. 10A) and would be the sequences RPLLGEM (SEQ ID NO:3), AKMEKEKLIS (SEQ ID NO: 4), KHSYTHMM (SEQ ID NO: 5), FFYTLPPLEKFI (SEQ ID NO: 6), GDSTFNK (SEQ ID NO: 7), NLYGSGMRS (SEQ ID NO: 8), LQYCTKLC (SEQ ID NO: 9), GSQSLTCM (SEQ ID NO: 10), NNFGPHI (SEQ ID NO: 11), TSLKIYGFRGIH (SEQ ID NO: 12), IIHECPFLTLS (SEQ ID NO: 13), RICYNKVA (SEQ ID NO: 14), and KYLTISRCN (SEQ ID NO: 15). It is believed that one or more of these sequences provide the functional characteristics of the protein Rp1-blb.
Furthermore, the invention provides a binding molecule directed at a nucleic acid according to the invention. For example, the Rpi-blb gene can be used for the design of oligonucleotides complementary to one strand of the DNA sequence as depicted in FIG. 7 and Table 2. Such oligonucleotides as provided herein are useful as probes for library screening, hybridisation probes for Southern/Northern analysis, primers for PCR, for use in a diagnostic kit for the detection of disease resistance and so on. Such oligonucleotides are useful fragments of an isolated or recombinant nucleic acid as provided herein, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae family with resistance against an oomycete fungus, or a functionally equivalent isolated or recombinant nucleic acid, in particular wherein said member comprises S. tuberosum or Lycopersicon esculentum. They can be easily selected from a sequence as depicted in FIG. 6 or part thereof. A particular point of recognition comprises the LRR domain as identified herein. Such a binding molecule according to the invention is used as a probe or primer, for example provided with a label, in particular wherein said label comprises an excitable moiety which makes it useful to detect the presence of said binding molecule.
The invention furthermore provides a method for selecting a plant or plant material or progeny thereof for its susceptibility or resistance to an oomycete infection comprising testing at least part of said plant or plant material or progeny thereof for the presence or absence of a nucleic acid, said nucleic acid encoding a gene product that is capable of providing a member of the Solanaceae family with resistance against an oomycete fungus, or for the presence of said gene product, said method preferably comprising contacting at least part of said plant or plant material or progeny thereof with a binding molecule according the invention and determining the binding of said molecule to said part. Said method is particularly useful wherein said oomycete comprises P. infestans, allowing to select plants or planting material for resistance against late blight, for example wherein said plant or material comprises S. tuberosum. It is believed that by the phylogenetic tree analysis as discussed above, proteins that are highly homologous to Rpi-blb and which would yield resistance against plant pathogens could be easily identified. An example for this is the detection of the three highly homologous proteins RGC1-blb, RGC3-blb and RGC4-blb, which have not yet been shown to yield resistance to P. infestans, but which are nevertheless believed to be involved in pathogen resistance in plants.
Also, the invention provides use of a nucleic acid or a vector or a cell or a substance or a binding molecule according to the invention in a method for providing a plant or its progeny with at least partial resistance against an oomycete infection, in particular wherein said oomycete comprises P. infestans especially wherein said plant comprises S. tuberosum, said method for providing a plant or its progeny with at least partial resistance against an oomycete infection comprising providing said plant or part thereof with a gene coding for a resistance protein or functional fragment thereof comprising a nucleic acid, said resistance protein being capable of providing a member of the Solanaceae family with resistance against an oomycete fungus, or providing said plant or part thereof with a nucleic acid or a vector or a cell or a substance according to the invention.
Furthermore, the invention provides an isolated S. bulbocastanum, or part thereof, such as a tuber or seed, susceptible to an oomycete infection caused by P. infestans. 
The invention is further described in the detailed description below.